Vaccine and process fob manufac



Reisaued'Dec. 1, 1953 VACCINE AND PROCESS FOR TUBING THE SAME musse Arden a. mums: and 1011118. Dick, rm Dodge, Iowa, asaignors to Fort Dodge Laboratories, Inc., Fort Dodge, Iowa, a corporation of Delaware N Brim.

Original m. min. dated inn-u 22, 1952, Serial No. 88,798, April 28, 1949. Application for reissue August 23, 1952, Serial No.

1s cum. (0|. m-so) Hatter enclosed in is, bracketl l: 1 appears in the original patent but forms no part of um cessful method of immunization of hogs against hog cholera which was developed by Dorset and others, depended upon the use of live hog cholera virus derived from an aimal sick with the disease. used simultaneously with an anti-serum against hog cholera virus. This method is dependent upon the use of varying amounts of serum and a more or less constant dose of virus. The variation in the dosage of serum depends upon the weight of the individual being immunized. Although the method confers immediate protection-against hog cholera active immunity is not strong for approximately two weeks. This, however; is not apparent because the passive iminunit'y carries the individual over until active immunity can be established.

.One outstanding disadvantage of this method is the fact that live virus is constantly seeded upon the premises, which is a potential source of hog cholera in susceptible animals. Furthermore, sometimes a false sense of security is had by the owner following vaccination in-which virus of low potency has been used and the resulting immunity is merely a passive impunity which may be lost in as few as twenty-one days. Nevertheless this method. from its beginning in the ear 1900's, is widely recognized and widely used to Recognizing the deficiencies of the above-described method of immunization, further investigations resulted in new methods seeking to obtainvaccines by a so-called attenuation of the virus. The proposed procedures of Boynton; Tilley. and others involving the use of chemicals ind heat generally result either in a type of attenuation where the virus is rendered incapable of growing when infected into a susceptible hog or actually destroys the hog cholera virus. instead of having a truly attenuated virus pable of reproducing to the point of insuring .a life-long immunity, prior vaccines depended upon the immunization of hogs against hog cholera on thebasis of the antigenic qualities ofthe virus. These vaccines do not give immediate protection but require at least seven days in whichto stimulate an active immunity and the resulting immunity has been found to be somewhat transient-in other words, theimmunity does not seem to last-and frequently hogs must be re-vaccinated after six months and at pcmatter printed in italics indicates the additions made by reissue.

This method cannot safely be used in conjunction with anti-hog cholera serum. nor has it been recommended for use within two weeks following the use of anti-hog cholera serum. It has also been found that swine so vaccinated are often hypersensitive to infection and may sicken from causes other than hog cholera during the immunization period.

The present invention successfully overcomes the deficiencies inherent in the prior methods briefly described since byv the use of the procedure hereinafter described. a rabbit-attenuated, swine-. propagated hog cholera virus product of concentrated strength is obtained which, in relatively small doses, is capable of conferring on swine a lasting immunity to hog cholera in contradistinction to the prior vaccines, and without the need of virulent hog cholera virus for full protection as heretofore required when relying upon the prior anti-hog cholera serum method of protection. By this invention-therefore, fully adequate protection to hog cholera is achieved and, in addition, the present-day danger of perpetuating hog cholera by seeding the virulent virus on the farm is completely avoided.

The attenuation of the virulent hog cholera virus is known to be capable of being brought about by'serial passage through rabbits. Attenuation is brought about by injecting hog cholera virus intravenously into rabbits. The .temperature of jthe rabbits is taken daily and, when a temperature rise is evident, the rabbits are sacrificed, their blood. or spleens or other viscera/pooled. finely ground if solids are used,

saline suspension and injected intravenously into a second R0 19 of rabbits. These, in turn, are sacrificed when a temperature rise is evident and the same procedure as above outlined is repeated.

40 Serial passage is thus carried out until the virus place, it is necessary to iniect'a portion of the macerated tissue suspension (if this is used) from each rabbit e into hog cholera susceptible swine. When attenuation is reached, the swine may show a fever but this is the only sign of illness. This can now be termed a rabbit-attenuated hog cholera virus. Apparently, the

virulent hog-cholera virus is modified by passing serially through rabbits from a disease-producing virus to one incapable of causing disease. The

riodic intervals thereafter to insure immunity. as virus is viable. however, and still retains an antiand a portion of this material is made into a i sane- 3 v genicity factor and, when injected into a susceptible hog,-generally stimulates it to produce an active immunity mainst hog cholera.

It has been found, however, that defibrinated rabbit blood or tissue suspension containing 5 rabbit-attenuated hog cholera virus is lacking in importantchara'cteristlcs. For the general imcentration of the attenuated virus in the blood I and tissues of the-rabbit has been found to be undesirably low. and therefore relatively large a amounts are required to obtain satisiactory 1mmunization. As a final factor leading away-from the utilisation oi rabbit tissue or blood'for immugeneral containing the attenuated virus are sepanization, the yield of immunizing material is low therefore a considerable number of animals are required. These' disadvantages obviously discovered that'swinm'yvhich were inoculatedwith rabbit-attenuatedwirus, built up a surprisingly high concentration or attenuated virus in their blood and tissues such as spleen, liver, kidney,v t

testes, etc; Moreover, it was also found that these]; materials with proper treatment'couldbe used 3 removed under aseptic conditions for procas a highly effective att'enua -virus'vaccine,

I since the virus-did not increase in virulence when injected into swine. Thus, instead 01 the low'y concentration, low-yield rabbit product, a highly concentrated swine-prosecuted vaccine is ob-, t F p v is used, is first defibrinated by known means and tained in much greater yields. In addition, this product is obviously less liable to produce shock 7 because of greater compatibility.

\ In accordance with the invention, rabbitattenuated hog cholera virus contained in tissue,

spleen or other. viscera in suspension or defibri nated rabbit blood is injected into a hog. UpOn the parenteral infection oi the rabbit-attenuated virus into swine, a rise in temperature is generally noted within threeto five days. This tem- 5 onset of fever, .the blood and/or any tissue or viscera will harbor the attenuated virus and thus I will constitute the source for obtaining the desired product which, when injected parenterally into susceptible swine, will stimulate those swine to V become actively immune to hog cholera. This product isnot only more compatible to swine than rabbit tissue but, more important, it contains a.

greater concentrationoi attenuated virus, requirin: the use of relatively small amounts for achisving the desired eflect. Furthermore, by carrying out the propagationcf attenuated virus in the yield of virus is greater because'of the com-- parative differences in side between rabbits and swine. I As a specific illustration oithe method for obtaining the product oi the invention, virulent,

hog'eholera virus is rendered bacteriologically' sterile and is injected intravenously into main Temperatures are taken twice'daily and, when a n temperature rise is noted, the rabbits are saerificed, their spleens pooled, macerated, and suspended indiluent such as physiological saline. A portion of this is then injected in a second group of rabbits. These'are sacrificed 7| 4 when a temperature rise is evident and the same procedure as above outlined is repeated. Generally. on about the thirteenth rabbit passage. attenuation is achieved. The rabbit-attenuated hog cholera virus is now ready for propagation in swine; Rabbit tissue-is finely ground, .suspended in physiologic'salt or other diluent and is'injected parenterally into cholera-susceptible swine. The dosage may vary in limits from about 54. cc. of a 1% spleen suspension-to about 10 cc. of a 10% spleen suspension in diluent, by weight, basedmn the entire composition. Within three to five days a temperature rise is observed, generally from about i02-105 E, this rise persisting irom about three to live days. On the second or third day from the onset oi i'ever, the animal ii sacrificed and the blood, tissues and viscera in rated for processing.

It may be pointed out that it is possible to obtain the desired product without sacrifice of the animal. For example, the hog may be bled. I a at intervals and the blood thus obtained may be used to prepare the product. As a further method, the spleen may be removed surgically under aseptic conditions, thus saving the hog.

Still further,.one may use male hogs, these being I injected .with rabbit-attenuated virus directly into the testicle. On the second or third day after the onset of fever, the testicles are surgi c ssing. Thus, this method also provides a good To obtain-the desired product, the blood, it this then rendered bacteriologically sterile by the addition or phenol or other similarly acting substance. If hog tissue is used, the tissue is finely ground and screened. The fine material may either be dried and later reconstituted or suspended in a diluent at the time of use if desired. or may-immediately be addedto a buii'ered isotonic solution or admixed with glycine, lactose or other suitable diluents. In addition, other active substances or excipients may be incorporated with the product, if desired. As a suitable 'compositiom' using either attenuated virus in deflbrinated hog blood or clear blood serumorfinelygroundhogtissueemaybemade up with crystal violet and glycerol in the proportions taught by Tilley in his Patent 2,369,267,

dated February 13, 1945.

with, about '5 held at ordinary .melbogspleenpreparedasinitxample as, no

' refrigerator temperature for at least one hundred days. Even longer times have been obtained when using about or more x of a baoteriostatic die. In the absence of these added agents, the attenuated virus vaccines must be used withina short time after its preparation for eifective results. Additionally, it is believed. that the use of added agents is effective in'preventing secondary infections to which the swine are attimes susceptible following their immunisation to bog cholera virus. Antibiotic, bacteriostatie or bactericidal agents and even gamma-globulin, either alone or in admixture.

140,000, 1400,000 and 14,000,000 four p ss new dilution. Simultaneously, each pig was injected subcutaneously in the axilla on the other side with 10 cc. of anti-hog cholera serum. Rectal temperatures of the pigs were taken daily for twenty-one days. During this period, only one pig in the 140,000 dilution showed any significant temperature rise.

In order to allow the passive immunity conferred by the serum to become dissipated, the resistance'oi' these pigs was challenged with 2 cc. of yirulent'hog cholera virus blood six weeks used in conjunction with the attenuatedhog 'chlorea virus for the combined purpose of immunising against hog cholera and protecting against secondary infections, coact to fully maintainthshsalthofthehogduring the timethat a solid immunity to hogeholera is being achieved.

Still another active agent which may be used in conjunction with the attenuated hog cholera virus is the well-knownanti-hog cholera serum hereinabove. As already indicated, prlorpractioeofimmunizinghogseonsistsof injectin'g anti-hog cholera serum along with aninjeetion of virulent hog cholera virus. if it is desired to use this serum, whether for achieving immediate temporary passive immunity to cholera or for preventing secondary infections, it has been found that excellent results may be attained without the use of virulent hog cholera virus by using the attenuated virus products of the invention together with the anti-hog cholera serum.

The following examples are further illustrative of the invention:

EXAMPLE I A sterile 10% suspension ofhog spleen containing rabbit-attenuated, swine-propagated hog cholera virus was prepared by grinding the spleen in a tissue grinder with physiologic saline until a homogenous suspension was prepared. I

, From this 10% spleen suspension, serial tenfold 1-1.0,000, 1-100, 000 and 14,000,000, four pigs per" dilution. Rectal temperatures of the pigs were taken daily for tweny-one days.

Duringthis period, four of four pigs injected with the 140,000 dilution, three of four pigs injected-with the 1400,000 dilution and two of after injection. Three pigs injected with the 14,000,000 dilution died after challenge. balance survived without evidence of cholera.

On this basis, it is concluded that the minimum immunizing dose of rabbit-attenuated, swinepropagated hog cholera virus when given simultaneously with 10 cc. of anti-hog cholera serum is in the range of 1 cc. of a 1400.000 to 14,000,000 dilution.

It should be noted that, while the above example indicates the more preferred method of separate injection of virus and serum, the antihog cholera may be admixed with the attenuated virus suspension for injection, if so desired. When both serum and attenuated virus are injected, the amount of each such should be selected so that the virus is not completely killed.- It has been found that approximately 545 oc.

of anti-hog cholera serum, when used together with approximately 1 cc. of attenuated virus prepared as described above, satisfactorily im- -munizes hogs against hog cholera and addition- .blood is more than adequate, while 10cc. of at- Having described our invention, what we claim I is as follows: I

1. In a process of preparing a vaccine for immunizing swine against hog cholera wherein a four pigs injected with label-1,000,000 dilution showed elevated temperatures.

Twenty-one days'after the iniection of the hog spleen dilutions, the resistance of all pigs was challenged by the injection of 2 cc. of virulent hog cholera virus blood. One pig which has received the 14,000,000 dilution died after challenge. All other pigs survived without evidence of cholera.

On this basis it is concluded that the minimum immunizing dose of the rabbit-attenuated, swineprop sfltedhog cholera virus is in the range of 1 cc. of a 1-1oo,ooo-m 14,090,000 dilution.

' miss an n in the axilla with 1 cc. of theserial diluvirulent strain of hog cholera virus is attenuated solely by about 13 to 15 serial rabbit trans-. fore, the improvement comprising propagating e'sired hog fractions and finally isolating a I, 1 hog cholera vaccine substance of hog origin con- The ' concentration,

tainingattenuated hog cholera virus in relatively high concentration.

2. A process of preparing a vaccine for immunizing swine against hog cholera, comprising the steps of attenuating a virulent strain of hog cholera. virus solely by about 13 to 15 serial rabbit transfers, isolating the rabbit substance containing attenuated hog cholera virus in relatively low concentration, concentrating said attenuated virus by injection of said rabbit substance into aims and, when a temperature rise above normal is obtained, bleeding the hog. treating the hog blood containing the virus in rel tile high concentration to remove fibrin therefrom, bacteriologically sterilizing the deflbrinated hog blood and'obtaining therefrom a vaccine comprising d8- nbrinated hog blood carrying attenuated hog cholera virus in a relatively high concentration 3.---A process of preparing a vaccine for im- .munizing swine against hog'cholera, comprising the steps oi attenuating a virulent strain of hog cholera virus solely by about 13 to 15 serial rabbit transfers, isolating the rabbit substance containing attenuated hog cholera virus in relatively low concentration, concentrating said attenuated virus by injection of said rabbit substance into a hogand, when a temperature riseabove normal is obtained, harvesting the hog spleen containing 7 the virus in relatively high concentration, finely grinding said hog spleen, screening the finely ground materialto remove undesired large particles and thereby obtaining a hog spleen tissue containing hog cholera virus in attenuated form and at a relatively high concentration.

4. A prccas of preparing a vaccine tor immunising swine against hog cholera, comprising the steps of attenuating a virulent strain of hog vii-us solely by about 13 to- 15 serial rabbit transfers, isolating the rabbit substance containing attenuated hog cholera virus in relatively low concentrating said attenuated the process of claim 5 [l] andcapable of being administered with a serum conferring immediate passive immunity. comprising hog substance containing attenuated hog cholera virus wherein 1 cc. of a greater dilution than 1:10.000 (hog substanceidiluent) is capable of conferring immunity to hogs from hog cholera disease.

7.- [6.] A hog cholera vaccine compositionv produced according to the process of claim 5 [1], comprising hog substance containing a relatively concentrated amount of rabbit-attenuated, swine-propagated hog cholera virus capable of conferring long. lasting immunity to hog cholera in swine and wherein to confer said immunity each cc. need not contain more than about 0.01 %-0.000l% of said hog substance.

8. [-1.] A hog cholera virus vaccine produced according to the process of claim 5 [1], comprising hog' substance containing attenuated but live hog cholera virus in a concentration measured by the standard that the vaccine is substantially capable of immunizing swine against hog cholera disease at a dilution, hog substancezdiluting medium, of substantially greater than 1:10,000 but not more than about 1':1,000,000.

9. In a process of preparinga vaccine for immunizing swine against hog cholera wherein a virulentstrai'n of hog cholera virus is attenudted, said attenuation being obtained solely by serial rabbit transfers, with attenuation of the virulence taking place after about 13 to 15 rabbit transfers, the improvement comprising propagating hog cholera attenuated virus in relatively low concentration through swine and securing a hog product containing attenuated virus in relalively concentrated form, by infection of the virus attenuated by rabbit transfers into a hog and, 'when a temperature rise above normal is virus by injection of said rabbit substance into 1 the testicles of a hog and; when a temperature rise above normal is obtained, surgically removing the testicles, comminuting the hog testicles to fine. particle size, screening oil undesired large particlcs or the hog tissue from the finely R und ate ial and thereby obtaining hog testicle tissue as a vaccine substance containing attenuated hog, cholera virus in a relatively high concentration.

5. In a process of preparing a vaccine for tm-i muniztng swine against hog cholera wherein a virulent strain of hog cholera virus is attenuated, uiid attenuation being obtained solely by serial rabbit transfers, with. attenuation of the virulence taking place after about 13 to 15 rabbtttranslers, th improvement comprising pr ting hog cholera attenuated virus through swine and securlnga hog product containing attenuated virus in concentrated form, by injection of thevirus 'sttenuflted by rabbit transfers into a hog and, a when a temperature rise above normal is obtained,

harvesting hog, subrtdnce containing the. virus in relatively high concentration, sold hog substance being selected from the group consisting of hog blood, hog spleen and hog testicle, treating said hog substance to remove undesired fractions obtained, harvesting the hog spleen containing the virus in relatively high concentration, finely cold -hog spleen, screening the finely ground material to remove undesired large particles and thereby obtaining a hog spleen tissue containing hog cholera, virus in attenuated form and at d-rel tively high concentration.

10. [9;] A hog cholera virus vaccine produced according to the process-oi" claim 9 [2], comprising hog tissue containing attenuatedbut live hog cholera virus in a concentration measured by the standard that the vaccine is substantially capable of immunizing swine against hog cholera disease at a dilution, hog substancezdiluting medium, of substantially greater than 1:10.000 but not more than about\1:1,000,000; said tissue carrying said attenuated virus being commingled with a bacteriostatic agent in an amount to provide eiifective bacteriostatic action.

11. In a process of preparing a vaccine for immunizing swine against hog cholera wherein j d virulent strain of hog cholera virus is attenof the group consisting of'tlbrin and unduly large particles incapable of passingthrough a fine screen, discarding said undesired hog fractions and finally isolating a hog cholera vdccine subwtcnce of hog origin containing attenuated hog cholera virus in relatively high concentration.

6. [5.] .A rabbit-attenuated. swine-propagated hog cholera virus vaccine produced according to noted, said dttenu tionbeingobtained solely by serial rabbit transfers, with attenuation of the vhulencetaking place after about 13 to 15 rabbit transfers, the improvement comprising propdgating hog cholera attenuated virus in relatively low concentration through swine and securing is hog product containing attenuated virus in relatively concentrated form, by injection of the virus ttenuatedby rabbit transfers into a hog and, when a temperature rise above normal is obtained, bleeding the hog, treating the hog blood containing the virus; in relatively high concentration to remove-fibrin therefrom, bacterlologlcgily sterilizing the deflbrindted hog sane 9 blood and obtaining therefrom a vaccine comprising deflbrinated hog blood carrying attenuated hog cholera virus in a relatively high concentration.

12. [8.] A hog cholera virus vaccine produced according to the process of claim 11 [3], comprising hog blood containing attenuated but live hog cholera virus in a concentration measured by the standard that the vaccine is substantially capable of immunizing swine against hog cholera disease at a dilution, hog substance:diluting medium, 01' substantially greater than 1:10,!)00 but not more than about 1:1,000,000; said hog substance carrying said attenuated virus being. oommingled with an antiseptic substance in an amount eflective to provide antiseptic action.

13. In a process of preparing a vaccine for immunizing swine against hog cholera wherein a virulent strain of hog cholera virus is attenuated, said attenuation being obtained solely by serial rabbit transfers, with attenuation of the virulence taking place generally after about 13 to 15 rabbit transfers (when attenuation is reached,'the swine may show a fever but this is the only sign of illness), the improvement comprising propagating hog cholera attenuated virus through swine and securing a hog product containing attenuated virus in concentrated form, by injection of the virus attenuated by rabbit transfers into a hog and, when a temperature rise above normal is obtained, harvesting hog substance containing the virus in relatively References Cited in the file 01' this patent or the original patent UNITED STATES PATENTS Number Name Date 1,246,059 Couret Nov. 13, 1917 1,595,377 Boynton Aug. 10, 1926 2,012,789 Kraybill Aug. 27, 1935 2,369,267 Tilley Feb. 13, 1945 2,518,978 Cox Aug. 15, 1950 OTHER REFERENCES Koprowski on Hog Cholera Virus in Rabbits, pages 178-183.

Baker on Hog Cholera Virus in Rabbits, pages 183-187.

Both of above in Proc. Soc. Exptl. Biol. 8: Med, vol. 63, No. 1, October 1946. 167-80.

Healy: "Attenuation of Hog Cholera Virus, in J. Infect. Dis. 19, P8888 588-571 (1918). 167-80. 

